1. Field of the Invention
The present invention relates to scanning systems for examining biological material and, in particular, to noise reduction in optical scanning systems having a laser to excite fluorescently tagged biological materials.
2. Related Art
Synthesized nucleic acid probe arrays, such as Affymetrix(copyright) GeneChip(copyright) synthesized probe arrays, have been used to generate unprecedented amounts of information about biological systems. For example, a commercially available GeneChip (copyright) array set from Affymetrix, Inc. of Santa Clara, Calif., is capable of monitoring the expression levels of approximately 6,500 murine genes and expressed sequence tags (EST""s). Experimenters can quickly design follow-on experiments with respect to genes, EST""s, or other biological materials of interest by, for example, producing in their own laboratories microscope slides containing dense arrays of probes using the Affymetrix(copyright) 417(trademark) or 427(trademark) Arrayers or other spotting devices. Analysis of data from experiments with synthesized and/or spotted probe arrays may lead to the development of new drugs and new diagnostic tools.
In some conventional applications, this analysis begins with the capture of fluorescent signals indicating hybridization of labeled target samples with probes on synthesized or spotted probe arrays. The devices used to capture these signals often are referred to as scanners. Due to the relatively small emission signals sometimes available from the hybridized target-probe pairs, the presence of background fluorescent signals, the high density of the arrays, variations in the responsiveness of various fluorescent labels, and other factors, care must be taken in designing scanners to properly acquire and process the fluorescent signals indicating hybridization. For example, U.S. Pat. No. 6,171,793 to Phillips, et al., hereby incorporated herein in its entirety for all purposes, describes a method for scanning probe arrays to provide data having a dynamic range that exceeds that of the scanner. Nonetheless, there is a continuing need to improve scanner design to provide more accurate and reliable fluorescent signals and thus provide experimenters with more sensitive and accurate data.
Systems, methods, and products to address these and other needs are described herein with respect to illustrative, non-limiting, implementations. Various alternatives, modifications and equivalents are possible. For example, certain systems, methods, and computer software products are described herein using exemplary implementations for analyzing data from arrays of biological materials produced by the Affymetrix(copyright) 417(trademark) or 427(trademark) Arrayer. Other illustrative implementations are referred to in relation to data from Affymetrix(copyright) GeneChip(copyright) probe arrays. However, these systems, methods, and products may be applied with respect to many other types of probe arrays and, more generally, with respect to numerous parallel biological assays produced in accordance with other conventional technologies and/or produced in accordance with techniques that may be developed in the future. For example, the systems, methods, and products described herein may be applied to parallel assays of nucleic acids, PCR products generated from cDNA clones, proteins, antibodies, or many other biological materials. These materials may be disposed on slides (as typically used for spotted arrays), on substrates employed for GeneChip(copyright) arrays, or on beads, optical fibers, or other substrates or media. Moreover, the probes need not be immobilized in or on a substrate, and, if immobilized, need not be disposed in regular patterns or arrays. For convenience, the term probe array will generally be used broadly hereafter to refer to all of these types of arrays and parallel biological assays.
In accordance with one preferred embodiment, a method is described that includes the steps of (1) directing an excitation beam to a plurality of pixel locations on a substrate; (2) determining one or more representative excitation values, each related to a value of the excitation beam as directed to at least one of the plurality of pixel locations; (3) detecting an emission signal having one or more emission values; (4) correlating each of the one or more emission values with one or more of the representative excitation values; (5) providing at least one excitation reference value; (6) comparing the excitation reference value to at least one representative excitation value, thereby generating a normalization factor; and (7) adjusting at least one emission value based, at least in part, on the normalization factor. One or more probes of a biological microarray may be disposed in relation to the substrate; for example, probes may be coupled to the substrate. The one or more probes may be disposed at different probe locations on a surface of the substrate. In some implementations, the substrate may include different polymer sequences coupled to the surface of the substrate. The different polymer sequences may include different oligonucleotide sequences, wherein each of the different polymer sequences is coupled in a different probe location of the surface. In some implementations, each of the probe locations has an area of one-hundredth of a square centimeter or less. Also, in some implementations, the substrate may include more than one thousand different ligands of known sequence collectively occupying an area of less than one square centimeter, the different ligands occupying different known locations within the area.
In accordance with further implementations of these preferred embodiments, step (2) includes directing the excitation beam to a dichroic mirror, and determining the representative excitation values based on a partial excitation beam that passes through the dichroic mirror. Also, the emission signal may arise from the direction of the excitation beam to the plurality of pixel locations. As used in this context, the word arise is intended to have a broad meaning so as to encompass various cause and effect relationships wherein the directing of the excitation beam causes or results in, directly or indirectly, the emission signal. As just one non-limiting example, the excitation beam may be a laser beam directed to a location on the substrate where fluorescently labeled receptors are disposed, and the emission signal may be a fluorescent signal resulting from excitation of those labeled receptors. In these and other implementations, step (4) may include spatially correlating the emission values with the representative excitation values. Also in these and other implementations, step (2) may include determining a first representative excitation value related to a power of the excitation beam as directed to a first of the plurality of pixel locations; step (3) may include detecting an emission value arising from the direction of the excitation beam to the first pixel location; and step (4) may include correlating the first emission value with the first representative excitation value. In accordance with various embodiments, the excitation reference value may be based, at least in part, on at least one of the one or more representative excitation values, and/or on a plurality of representative excitation values related to values of the excitation beam as directed to pixel locations in one or more scan lines.
The method, in accordance with some embodiments, may further include the step of (8) filtering the representative excitation values to provide one or more filtered representative excitation values. In these embodiments, the excitation reference value is based, at least in part, on at least one of the one or more filtered representative excitation values. In these and other embodiments, the excitation reference value may be based, at least in part, on a measured calibration value and/or on a predetermined specification value.
In accordance with yet other embodiments, a system is described for processing an emission signal having one or more emission values. The system includes an excitation signal generator that provides an excitation signal having one or more representative excitation values representative of an excitation beam. The system also has an excitation reference provider that provides at least one excitation reference value; a normalization factor generator that compares the excitation reference value to at least one representative excitation value, thereby generating a normalization factor; and a comparison processor that adjusts at least one emission value corresponding to the at least one representative excitation value based, at least in part, on the normalization factor. The excitation reference value may be determined based at least in part on a low-frequency component of the excitation signal. The at least one representative excitation value may include, as examples, an instantaneous analog value or a sampled digital value. In some implementations, the comparison processor adjusts the at least one emission value corresponding to the at least one representative excitation value based on multiplying or dividing the emission value by the normalization factor. The excitation signal may include a laser signal, and the emission signal may include a fluorescent signal resulting from excitation of a fluorophore by the laser signal.
In accordance with a further embodiment, a method is described for processing an emission signal having one or more emission values. The method includes providing at least one excitation reference value; comparing the excitation reference value to at least one excitation value, thereby generating a normalization factor; and adjusting at least one emission value corresponding to the at least one excitation value based, at least in part, on the normalization factor.
A scanning system is described in accordance with some embodiments. The system includes one or more excitation sources that generate one or more excitation beams; an excitation signal generator that provides an excitation signal having one or more representative excitation values representative of the excitation beam; an excitation reference provider that provides at least one excitation reference value; a normalization factor generator that compares the excitation reference value to at least one representative excitation value, thereby generating a normalization factor; and a comparison processor that adjusts at least one emission value corresponding to the at least one representative excitation value based, at least in part, on the normalization factor. The scanning system may include a processor and a memory unit, wherein the normalization factor generator includes a set of normalization factor generating instructions stored in the memory unit and executed in cooperation with the processor. The comparison processor may also include a set of comparison processing instructions stored in the memory unit and executed in cooperation with the processor.
A computer program product is described with respect to other embodiments. The product includes a set of normalization factor generating instructions stored in a memory unit of a computer and executed in cooperation with a processor of the computer. These instructions are constructed and arranged to compare at least one excitation reference value to at least one representative excitation value, thereby generating a normalization factor. The product also includes a set of comparison processing instructions stored in the memory unit and executed in cooperation with the processor, constructed and arranged to adjust at least one emission value corresponding to the at least one representative excitation value based, at least in part, on the normalization factor. The at least one emission value results from excitation of a labeled receptor at a probe location of a probe array.
A method for analyzing molecules is described with respect to some embodiments. The method includes (1) directing an excitation beam to a plurality of pixel locations on a surface having a plurality of probe locations, each probe location including one or more probe molecules; (2) determining one or more representative excitation values, each related to a value of the excitation beam as directed to at least one of the plurality of pixel locations; (3) detecting an emission signal having one or more emission values; (4) correlating each of the one or more emission values with one or more of the representative excitation values; (5) providing at least one excitation reference value; (6) comparing the excitation reference value to at least one representative excitation value, thereby generating a normalization factor; (7) adjusting at least one emission value based, at least in part, on the normalization factor; and (8) analyzing at least one probe location based, at least in part, on the at least one adjusted emission value.
The above embodiments and implementations are not necessarily inclusive or exclusive of each other and may be combined in any manner that is non-conflicting and otherwise possible, whether they be presented in association with a same, or a different, embodiment or implementation. The description of one embodiment or implementation is not intended to be limiting with respect to other embodiments and/or implementations. Also, any one or more function, step, operation, or technique described elsewhere in this specification may, in alternative implementations, be combined with any one or more function, step, operation, or technique described in the summary. Thus, the above embodiment and implementations are illustrative rather than limiting.